This invention relates generally to microfabricated extraction systems and methods for separating analytes from streams containing other constituents by differential transport principles such as diffusion and applied fields. The invention is useful, for example, for processing blood to separate a stream containing smaller particles such as albumin molecules from a stream containing cells. In another aspect this invention relates generally to microsensors and methods for analyzing the presence and concentration of small particles in streams containing both these small particles and larger particles by diffusion principles, and is useful, for example, for analyzing blood to detect the presence of small particles such as hydrogen, sodium or calcium ions in a stream containing cells.
In Maxwell""s famous gedanken (thought) experiment, a demon operates a door between two boxes of gas at the same temperature. The demon sorts the molecules, keeping the faster molecules in one box and the slower in the other, violating the basic laws of thermodynamics. This paradox has since been resolved in many different ways. Leff, H. S. and Rex, A. F. (1990), xe2x80x9cResource letter md-1: Maxwell""s demon,xe2x80x9d Am. J. Physics 58:201-209.
A similar arrangement can be used to separate particles. Consider a mixture of particles of two different sizes suspended in water in one box and pure water in the other. If the demon opens and closes the door between the boxes quickly enough so that none of the larger particles have time to diffuse through the doorway, but long enough so that some of the smaller particles have enough time to diffuse into the other box, some separation will be achieved.
Recently two experiments have been done where a spatially asymmetric potential is periodically applied in the presence of a number of Brownian particles. Faucheux, L. S., et al. (1995), xe2x80x9cOptical thermal ratchet,xe2x80x9d Physical Rev. Letters 74:1504-1507; Rousselet, J., et al. (1994), xe2x80x9cDirectional motion of Brownian particles induced by a periodic asymmetric potential,xe2x80x9d Nature 370:446-448. This has been shown to lead to a directed motion of the particles at a rate depending on the diffusion coefficient. One experiment (Rousselet, J., et al. (1994), xe2x80x9cDirectional motion of Brownian particles induced by a periodic asymmetric potential,xe2x80x9d Nature 370:446-448) used microfabricated electrodes on a microscope slide to apply an electric field for the potential. This idea is also the subject of European Patent Publication 645169 of Mar. 29, 1995, for xe2x80x9cSeparation of particles in a fluid using a saw-tooth electrode and an intermittent excitation field,xe2x80x9d Adjari, A., et al. The other experiment (Faucheux, L. S., et al. (1995), xe2x80x9cOptical thermal ratchet,xe2x80x9d Physical Rev. Letters 74:1504-1507) used a modulated optical tweezer arrangement.
Chemical analysis of biological samples is constrained by sample size. Withdrawing a few milliliters of blood from an adult may have little effect, but repeating this procedure every hour or even withdrawing this amount once from an infant can significantly alter the health of the subject. For these reasons, a miniaturized blood analysis system would be useful. Furthermore, while many sophisticated tests that have great importance for critical care can be performed in major hospital laboratories, a substantial impact could be made on the practice of emergency medicine if some key tests could be performed on the patient at the site of injury. For some assays it is vital to make measurements in the absence of red blood cells, so some form of separation of cells from plasma is required.
Diffusion is a process which can easily be neglected at large scales, but rapidly becomes important at the microscale. The average time t for a molecule to diffuse across a distance d is 2t=d2/D where D is the diffusion coefficient of the molecule. For a protein or other large molecule, diffusion is relatively slow at the macroscale (e.g. hemoglobin with D equal to 7xc3x9710xe2x88x927cm2/s in water at room temperature takes about 106 seconds (ten days) to diffuse across a one centimeter pipe, but about one second to diffuse across a 10 xcexcm channel).
Using tools developed by the semiconductor industry to miniaturize electronics, it is possible to fabricate intricate fluid systems with channel sizes as small as a micron. These devices can be mass-produced inexpensively and are expected to soon be in widespread use for simple analytical tests. See, e.g., Ramsey, J. M. et al. (1995), xe2x80x9cMicrofabricated chemical measurement systems,xe2x80x9d Nature Medicine 1:1093-1096; and Harrison, D. J. et al (1993), xe2x80x9cMicromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip,xe2x80x9d Science 261:895-897.
Miniaturization of analytic instruments is not a simple matter of reducing their size. At small scales different effects become important, rendering some processes inefficient and others useless. It is difficult to replicate smaller versions of some devices because of material or process limitations. For these reasons it is necessary to develop new methods for performing common laboratory tasks on the microscale.
Devices made by micromachining planar substrates have been made and used for chemical separation, analysis, and sensing. See, e.g., Manz, A. et al. (1994), xe2x80x9cElectroosmotic pumping and electrophoretic separations for miniaturized chemical analysis system,xe2x80x9d J. Micromech. Microeng. 4:257-265.
Field flow fractionation devices involve particle size separation using a single inlet stream. See, e.g. Giddings, J. C., U.S. Pat. No. 3,449,938, Jun. 17, 1969, xe2x80x9cMethod for Separating and Detecting Fluid Materialsxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,147,621, Apr. 3, 1979, xe2x80x9cMethod and Apparatus for Flow Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,214,981, Jul. 29, 1980, xe2x80x9cSteric Field-Flow Fractionationxe2x80x9d; Giddings, J. C. et al., U.S. Pat. No. 4,250,026, Feb. 10, 1981, xe2x80x9cContinuous Steric FFF Device for The Size Separation of Particlesxe2x80x9d; Giddings, J. C. et al. (1983), xe2x80x9cOutlet Stream Splitting for Sample Concentration in Field-Flow Fractionation,xe2x80x9d Separation Science and Technology 18:293-306; Giddings, J. C. (1985), xe2x80x9cOptimized Field-Flow Fractionation System Based on Dual Stream Splitters,xe2x80x9d Anal. Chem. 57:945-947; Giddings, J. C., U.S. Pat. No. 4,830,756, May 16, 1989, xe2x80x9cHigh Speed Separation of Ultra-High Molecular Weight Polymers by Hyperlayer Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,141,651, Aug. 25, 1992, xe2x80x9cPinched Channel Inlet System for Reduced Relaxation Effects and Stopless Flow Injection in Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,156,039, Oct. 20, 1992, xe2x80x9cProcedure for Determining the Size and Size Distribution of Particles Using Sedimentation Field-Flow Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,193,688, Mar. 16, 1993, xe2x80x9cMethod and Apparatus for Hydrodynamic Relaxation and Sample Concentration in Field-Flow Fraction Using Permeable Wall Elementsxe2x80x9d; Caldwell, K. D. et al., U.S. Pat. No. 5,240,618, Aug. 31, 1993, xe2x80x9cElectrical Field-Flow Fractionation Using Redox Couple Added to Carrier Fluidxe2x80x9d; Giddings, J. C. (1993), xe2x80x9cField-Flow Fractionation: Analysis of Macromolecular, Colloidal and Particulate Materials,xe2x80x9d Science 260:1456-1465; Wada, Y. et al., U.S. Pat. No. 5,465,849, Nov. 14, 1995, xe2x80x9cColumn and Method for Separating Particles in Accordance with Their Magnetic Susceptibilityxe2x80x9d; Yue, V. et al. (1994), xe2x80x9cMiniature Field-Flow Fractionation Systems for Analysis of Blood Cells,xe2x80x9d Clin. Chem. 40:1810-1814; Afromowitz, M. A. and Samaras, J. E. (1989), xe2x80x9cPinch Field Flow Fractionation Using Flow Injection Techniques,xe2x80x9d Separation Science and Technology 24(5 and 6):325-339.
Thin-channel split flow fractionation (SPLITT) technology also provides particle separation in a separation cell having a thin channel. A field force is exerted in a direction perpendicular to the flow direction. Particles diffuse or are otherwise transported from a particle-containing stream across a transport stream to a particle-free stream. The device for operating the process is generally fabricated from glass plates with Teflon sheets used as spacers to form the channels. The channel depth can therefore be no smaller than the spacers, which are generally about 100 to 120 xcexcm thick. See, e.g., Giddings, J. C., U.S. Pat. No. 4,737,268, Apr. 12, 1988, xe2x80x9cThin Channel Split Flow Continuous Equilibrium Process and Apparatus for Particle Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 4,894,146, Jan. 16, 1990, xe2x80x9cThin Channel Split Flow Process and Apparatus for Particle Fractionationxe2x80x9d; Giddings, J. C., U.S. Pat. No. 5,093,426, Aug. 13, 1991, xe2x80x9cProcess for Continuous Particle and Polymer Separation in Split-Flow Thin Cells Using Flow-Dependent Lift Forcesxe2x80x9d; Williams, P. S. et al. (1992), xe2x80x9cContinuous SPLITT Fractionation Based on a Diffusion Mechanism,xe2x80x9d Ind. Eng. Chem. Res. 31:2172-2181; and Levin, S. and Tawil, G. (1993), xe2x80x9cAnalytical SPLITT Fractionation in the Diffusion Mode Operating as a Dialysis-like System Devoid of Membrane. Application to Drug-Carrying Liposomes,xe2x80x9d Anal. Chem. 65:2254-2261.
An object of this invention is to provide a microfabricated extraction system utilizing differential transport principles in which an analyte can be extracted, detected and quantified.
The advantages, as disclosed herein, of diffusion separation devices on the microscale, e.g., having channel depths no greater than about 100 xcexcm, do not appear to have been recognized in the prior art. See, e.g., Kittilsand, G. and Stemme, G. (1990), Sensors and Actuators A21-A23:904-907, and Wilding, P. et al. (1994), J. Clin. Chem. 40:43-47. None of the foregoing publications describe a channel system capable of analyzing small particles in very small quantities of sample containing larger particles, particularly larger particles capable of affecting the indicator used for the analysis. No devices or methods using indicator streams within the cell system are described.
All publications, patents and patent applications referred to herein are hereby incorporated by reference.
Microfluidic devices allow one to take advantage of diffusion as a rapid separation mechanism. Flow behavior in microstructures differs significantly from that in the macroscopic world. Due to extremely small inertial forces in such structures, practically all flow in microstructures is laminar. This allows the movement of different layers of fluid and particles next to each other in a channel without any mixing other than diffusion. On the other hand, due to the small lateral distances in such channels, diffusion is a powerful tool to separate molecules and small particles according to their diffusion coefficients, which are usually a function of their size.
In one aspect, this invention provides an extraction method and device distinguished from conventional filtration techniques and devices in possessing advantages of size, production economy, integrability with micro chemical analysis systems, low power consumption, and which may be operated in either a sample-to-sample or continuous processing mode. The device is particularly well suited to integration with microfabricated chemical analysis systems in which, for example, a preferred embodiment provides a microfabricated extraction device or system capable of providing a diluted plasma product having a volume ranging from picoliters to nanoliters starting from samples as small as a microliter of whole blood, with a comparable extraction stream volume.
The extraction system is useful as an element in an integrated system of microfluidic and detection elements (such as optical detectors) for tests of medical interest on blood, and also has applications in many other areas of analytical chemistry. In a preferred embodiment useful for blood analysis, the device allows for the extraction of plasma constituents from whole blood, thereby producing a cell-free fluid stream for subsequent analysis.
The microfabricated extraction system of this invention in simplest concept is illustrated by a diffusion extraction device comprising microchannels in the shape of an xe2x80x9cHxe2x80x9d. A mixture of particles suspended in a sample stream enters the extraction channel (the crossbar of the xe2x80x9cHxe2x80x9d) from one of the arms, e.g. the top left, and an extraction stream (a dilution stream) enters from the bottom left. The two streams flow together in the extraction channel; however, due to the small size of the channels, the flow is laminar and the streams do not mix. The sample stream exits as by-product stream at the upper right and the extraction stream exits as product stream from the lower right. While the streams are in parallel laminar flow in the extraction channel, particles having a greater diffusion coefficient (smaller particles such as albumin, sugars and small ions) have time to diffuse into the extraction stream, while the larger particles (e.g. blood cells) remain in the sample stream. Particles in the exiting extraction stream (now called the product stream) may be analyzed without interference from the larger particles.
In this patent application, the flow direction of a channel is called its length (L). The channel dimension in the direction of particle transport at right angles to the length (L) is called its depth (d). The third channel dimension at right angles to both the length and depth is called its width (w). The depth (d) is therefore perpendicular to the plane of interface of the sample and extraction streams. Table 1 lists other abbreviations used herein.
The length of the extraction channel and the extraction channel flow velocity are key parameters determining the amount of time the particles have to diffuse into the extraction stream. The particles in the case described above are differentially transported from the sample stream to the extraction stream using diffusion as the transport mechanism. Other means for effecting differential transport of the desired particles can also be used. The term xe2x80x9cdifferential transportxe2x80x9d means that a portion of the desired particles are transported from the sample stream into the extraction stream to the substantial exclusion of the undesired particles. For example, magnetic, electrical or other forces can be applied across the extraction stream, temperature gradients can be used, or absorbent or adsorbent materials such as antibodies can be added to the extraction stream to capture the desired particles.
The microfabricated device of this invention for extracting desired particles from a sample stream containing said particles comprises: a sample stream inlet; an extraction stream inlet; an extraction channel having an aspect ratio (channel width to depth) less than 50 in fluid communication with said sample stream inlet and said extraction stream inlet for receiving a sample stream from said sample stream inlet in parallel laminar flow with an extraction stream from said extraction stream inlet; a by-product stream outlet in fluid communication with said extraction channel for receiving a by-product stream comprising at least a portion of said sample stream from which desired particles have been extracted; and a product stream outlet in fluid communication with said extraction channel for receiving a product stream comprising at least a portion of said extraction stream and comprising desired particles extracted from said sample stream.
In this extraction embodiment, the sample stream and extraction stream inlets and the by-product stream and product stream outlets may comprise channels, reservoirs, ports, or other containers. The sample stream inlet is designed to receive a sample stream containing xe2x80x9cdesired particles,xe2x80x9d i.e. particles it is desired to extract so that their presence may be detected. The sample stream also includes other particles which are not extracted, termed xe2x80x9cundesired particlesxe2x80x9d herein. These undesired particles include particles which might interfere with the detection of the desired particles. In a preferred embodiment, the sample stream comprises whole blood. The desired particles may be albumin or other blood plasma components, and the undesired particles are blood cells. The device is especially useful for obtaining cell-free plasma from whole blood. Other fluids for which the present invention is useful include solutions or suspensions of DNA fragments of different lengths, or proteins of varying sizes. Sample streams useful in the practice of this invention include fermentation broths, raw sewage, liquefied food samples, soil samples and biological fluids such as sputum, urine, and cerebral spinal fluid.
The diffusion analysis embodiment of this invention (also referred to herein as the xe2x80x9cT-sensorxe2x80x9d) provides a channel cell system for detecting the presence of analyte particles in a sample stream also comprising larger particles comprising: a laminar flow channel; at least two inlet means in fluid connection with said laminar flow channel for respectively conducting into said laminar flow channel (1) an indicator stream, said indicator stream preferably comprising an indicator substance, for example, a pH-sensitive dye, which indicates the presence of said analyte particles by a detectable change in property when contacted with said analyte particles, and (2) said sample stream; wherein said laminar flow channel has a depth sufficiently small to allow laminar flow of said streams adjacent to each other and a length sufficient to allow analyte particles to diffuse into said indicator stream to the substantial exclusion of said larger particles in said sample stream to form a detection area; and outlet means for conducting said streams out of said laminar flow channel, preferably to form a single mixed stream.
The channel cell system of a preferred embodiment of the diffusion analysis embodiment of this invention comprises channel grooves in the form of a xe2x80x9cTxe2x80x9d or a xe2x80x9cYxe2x80x9d having a central trunk and two branches etched into the surface of a silicon microchip, which surface is thereafter covered with a glass sheet. The central groove is formed of the trunk of the xe2x80x9cTxe2x80x9d or xe2x80x9cYxe2x80x9d, and the branches are the inlet means in fluid connection with the laminar flow channel for respectively conducting the sample and indicator streams into the laminar flow channel.
In the simplest embodiment of this diffusion analysis invention, a single indicator stream and a single sample stream are used; however, the methods and devices of this invention may also use multiple sample and/or indicator streams, and reference or calibration streams, all in laminar flow with each other. The preferred embodiments of this diffusion analysis invention utilize liquid streams, although the methods and devices are also suitable for use with gaseous streams. The term xe2x80x9cfluid connectionxe2x80x9d means that fluid flows between the two or more elements which are in fluid connection with each other.
This invention farther provides a microfluidic system comprising a plurality of inlets; means for controlling fluid flow through at least one of said inlets connected with said inlet; a laminar flow channel in fluid communication with said inlets having an aspect ratio (w/d) less than 50; and at least one outlet in fluid communication with said laminar flow channel. Preferably the system also comprises means for controlling fluid flow through said outlet. The microfluidic system may have a plurality of (two or more) outlets, and preferably has means for controlling fluid flow through at least one of said outlets connected with that outlet. In one embodiment of this invention, the system has two inlets. It may have two outlets, at least three, or at least four outlets. In one embodiment it has at least six inlets and at least six outlets.
Also provided is a microfluidic system comprising a plurality of inlets; means for controlling fluid flow through at least one of said inlets connected with said inlet; a laminar flow channel in fluid communication with said inlets; at least three outlets in fluid communication with said laminar flow channel; and means for controlling fluid flow through at least one of said outlets connected with said outlet. In one embodiment, the system has at least four outlets, e.g., at least six inlets and at least six outlets.
Means for controlling fluid flow may be connected to all inlets, all outlets, all but one outlet, all but one inlet, and in a preferred embodiment are connected to all inlets and all but one outlet, or all outlets and all but one inlet. The means for controlling fluid flow may be any means known to the art, including pressure control means such as columns of water, electroendoosmotic forces, optical forces, gravitational forces and surface tension forces.
The laminar flow channel is designed to contain at least a first and second fluid stream in side-by-side laminar flow. Preferably the channel is long enough such that particles contained in one of the fluid stream can diffuse into the second stream for separation or detection.
Further provided herein is a method for creating a fluid interface between two or more streams flowing within a microfluidic channel comprising: simultaneously flowing said streams into a laminar flow channel having an aspect ratio (w/d) less than 50; and allowing said streams to flow in side-by-side laminar flow within said channel. The streams may be of equal volume and/or flow rate or unequal volume and/or flow rate. In the latter case, where a greater volume of one stream is flowed into the one stream than the other, or a lesser volume is allowed to exit the channel from one stream than the other, a fluid barrier can be formed between the streams. The streams may comprise a particle-containing stream and a particle-receiving stream, such that particles contained in the particle-containing stream are allowed to diffuse into the particle-receiving stream.
Further provided is a microfluidic device comprising: a microfluidic channel having a first end and a second end; and means for simultaneously introducing at least two fluids into said first end of said channel.
The devices of this invention preferably have no internal structures, i.e. structures such as splitters within the channel, not including outlet port configurations, which would interfere with the parallel laminar flow of streams therein.
The term xe2x80x9cdetectionxe2x80x9d as used herein means determination that a particular substance is present. Typically, the concentration of a particular substance is determined. The methods and apparatuses of this invention can be used to determine the concentration of a substance in a sample stream.
The input streams of this invention include a sample stream containing particles to be extracted, detected or analyzed. In the separation embodiment, a second input stream is referred to as the extraction or dilution stream. In the diffusion analysis embodiment, a second input stream is referred to as the indicator stream. In the separation embodiment, the laminar flow channel is sometimes referred to as the extraction channel, and in the diffusion analysis embodiment the laminar flow channel is sometimes referred to as the diffusion channel.
One preferred embodiment entails the incorporation in the extraction or indicator stream of an adsorbent material such as a receptor with specificity for the desired ligand particles, onto an effectively non-diffusing substrate, such as plastic beads or high molecular weight polymers. Another preferred embodiment utilizes an effectively non-diffusing absorbent particulate material with specificity for the desired particles. Such materials are considered xe2x80x9ceffectively non-diffusingxe2x80x9d when they do not diffuse into the sample stream, or do not diffuse into the sample stream in quantities large enough to interfere with detection of the undesired particles in the by-product stream. In the absorbent embodiment, desired particles are absorbed within the effectively non-diffusing absorbing particulate material, whereas in the adsorbent embodiment, the desired particles attach to the surface of the effectively non-diffusing substrate plastic beads or to ligands attached thereto. Numerous suitable ligands for desired particles in the adsorbent/absorbent embodiment are known to the art, and specific teachings relative to these techniques are disclosed in application Ser. No. 08/876,038 filed Jun. 14, 1996, now U.S. Pat. No. 5,971,158 issued Oct. 26, 1999.
In the diffusion analysis embodiment of this invention, the channel cell system of this invention may comprise external detecting means for detecting changes in an indicator substance carried within the indicator stream as a result of contact with analyte particles. Detection and analysis is done by any means known to the art, including optical means, such as optical spectroscopy, and other means such as absorption spectroscopy or fluorescence, by chemical indicators which change color or other properties when exposed to the analyte, by immunological means, electrical means, e.g. electrodes inserted into the device, electrochemical means, radioactive means, or virtually any microanalytical technique known to the art including magnetic resonance techniques, or other means known to the art to detect the presence of an analyte such as an ion, molecule, polymer, virus, DNA sequence, antigen, microorganism or other factor. Preferably optical or fluorescent means are used, and antibodies, DNA sequences and the like are attached to fluorescent markers.
The term xe2x80x9cparticlesxe2x80x9d refers to molecules, cells, large molecules such as proteins, small molecules comprised of one or several atoms, and ions. The particles may be suspended or dissolved in the stream. The term xe2x80x9cstreamxe2x80x9d refers to a carrier fluid such as water or other liquid, air or other gas, containing desired and/or undesired particles. The term xe2x80x9cparticlesxe2x80x9d as used herein does not include the molecules of the carrier stream.
The term xe2x80x9cextractionxe2x80x9d refers to the separation of at least a portion, i.e. a detectable portion, of desired particles from the sample stream to the substantial exclusion of undesired particles. It is recognized that very small amounts of undesired particles may be transported into the extraction stream; however, the presence of such undesired particles will be minimized such that they do not interfere with detection or subsequent processing of the streams containing the desired particles.
The methods of this invention are designed to be carried out such that all flow is laminar. In general, this is achieved in a device comprising microchannels of a size such that the Reynolds number for flow within the channel is below about 1, preferably below about 0.1. The term xe2x80x9claminar flowxe2x80x9d of two streams means stable, side-by-side, non-recirculating, flow of two streams without mixing. There are no zones of recirculation, and turbulence is negligible.
The input or sample stream may be any stream containing particles of the same or different size, for example blood or other body fluid, contaminated drinking water, contaminated organic solvents, urine, biotechnological process samples, e.g. fermentation broths, and the like. The particles to be separated, or analyzed (xe2x80x9cthe analytexe2x80x9d), may be any smaller particles in the input stream capable of diffusing into the extraction or indicator stream in the device, e.g. hydrogen, calcium or sodium ions, proteins, e.g. albumin, organic molecules, drugs, pesticides, and other particles. In a preferred embodiment when the sample stream is whole blood, small ions such as hydrogen and sodium diffuse rapidly across the channel, whereas larger particles such as those of large proteins, blood cells, etc. diffuse slowly. Preferably the particles to be separated or analyzed are no larger than about 3 micrometers, more preferably no larger than about 0.5 micrometers, or are no larger than about 1,000,000 MW, and more preferably no larger than about 50,000 MW.
The diffusion analysis system includes an indicator stream introduced into one of the inlet means comprising a liquid carrier which may contain substrate particles such as polymers or beads having an indicator substance immobilized thereon. The indicator substance is preferably a substance which changes in fluorescence or color in the presence of analyte particles, such as a dye, enzymes, and other organic molecules that change properties as a function of analyte concentration. The term xe2x80x9cindicator substancexe2x80x9d is also used to refer to polymeric beads, antibodies or the like having dyes or other indicators immobilized thereon. It is not necessary that the indicator stream comprise an indicator substance when detection means such as those directly detecting electrical, chemical or other changes in the indicator stream caused by the analyte particles are used. The system may also include an analyte stream comprising substrate particles such as polymer beads, antibodies and the like on which an indicator substance is immobilized. The liquid carrier can be any fluid capable of accepting particles diffusing from the feed stream and containing an indicator substance. Preferred indicator streams comprise water and isotonic solutions such as salt water with a salt concentration of about 10 mM NaCl, KCl or MgCl, or organic solvents like acetone, isopropyl alcohol, ethanol, or any other liquid convenient which does not interfere with the effect of the analyte on the indicator substance or detection means.
In the devices of this invention, the streams may be separated at the end of the conduit at any arbitrary location by precise regulation of the exit flow rate of the outlets, something which is not possible at higher Reynolds numbers not satisfying the non-recirculating and non-turbulent criteria.
In the separation embodiment of this invention, the extraction stream inlet is designed to receive an extraction stream capable of accepting desired particles when in laminar flow contact with the sample stream. The extraction stream can be any fluid capable of accepting particles being transported from the sample stream. Preferred extraction streams are water and isotonic solutions such as physiological saline. Other useful extractant streams comprise organic solvents such as acetone, isopropyl alcohol, supercritical carbon dioxide or ethanol. Air and other gases may also be used as sample and extraction streams.
In the separation embodiment, the by-product stream comprises the sample stream from which a portion of the desired particles have been extracted and may or may not, as discussed below, be comprised of a fraction of the extraction stream into which desired particles have been conveyed from the sample stream. The by-product stream outlet is designed to conduct the by-product stream (composed of the sample stream and perhaps a portion of the extraction stream) that is removed from the extraction channel to disposal, recycle, or other system component, for further processing.
In this separation embodiment, the product stream comprises at least a portion of the extraction stream into which desired particles have been extracted. The product stream outlet, which as stated above, may comprise a product stream channel, is designed to conduct the product stream containing a detectable quantity of desired particles to a detection or further processing area or system component. A sufficient quantity of the extraction stream must be present in the product stream, comprising a sufficient quantity of desired particles, such that the presence of the desired particles is detectable in the product stream by means known to the art. The product stream may be conducted to a reservoir chamber, or other device where it may be further treated, e.g. by mixing, separating, analyzing, heating or otherwise processing, for example as disclosed in Wilding, P., et al. U.S. Pat. No. 5,304,487 issued Apr. 19, 1994, incorporated herein by reference.
The channel cell may be fabricated by microfabrication methods known to the art, e.g. as exemplified herein, a method comprising forming channels in a silicon microchip, such as by etching grooves into the surface of the silicon microchip and placing a glass cover over the surface. Precision injection molded plastics may also be used for fabrication. The term xe2x80x9cmicrofabricatedxe2x80x9d refers to devices capable of being fabricated on silicon wafers readily available to those practicing the art of silicon microfabrication and having the feature sizes and geometries producible by such methods as LIGA, thermoplastic micropattern transfer, resin based microcasting, micromolding in capillaries (MIMIC), wet isotropic and anisotropic etching, laser assisted chemical etching (LACE), and reactive ion etching (RIE), or other techniques known within the art of microfabrication. In the case of silicon microfabrication, larger wafers will accommodate a plurality of the devices of this invention in aplurality of configurations. A few standard wafer sizes are 3xe2x80x3, 4xe2x80x3, 6xe2x80x3, and 8xe2x80x3. Application of the principles presented herein using new and emerging microfabrication methods is within the scope and intent of the claims hereof.
The inlets need only be sized large enough to conduct the streams into parallel laminar flow, e.g., the device may comprise channels less than or equal to about 5 mm in length, less than about 100 micrometers in depth and less than or equal to 5 mm in width. The outlets may similarly be minimal in size, comprising channels with dimensions as stated above for the inlets. These inlets and outlets may be as long, deep and wide as required by the system of which they are a part, however, they preferably have a volume less than about 2.5 microliters to accommodate small sample sizes.
The width and depth of the inlet and outlet channels must be large enough to allow passage of the undesired particles, preferably anywhere between about 2 or 3 times the diameter of the undesired particles in the sample stream and less than or equal to about 5 mm. Particle sizes range from one or a few xc3x85 for small organic and inorganic molecules and ions to about 0.01 micrometers in depth for proteins, to about 0.1-1 micrometers for flexible long-chained molecules, to about 8 micrometers for red blood cells, to about 15 micrometers for most white blood cells, and up to about 25 micrometers for some white blood cells. The laminar flow channel must additionally be large enough to allow passage of particles used in the extraction or indicator stream, such as adsorbent or absorbent particles, and is preferably between about 2 or 3 times the diameter of such particles and less than or equal to 5 mm. The laminar flow channel is most preferably less than 100 micrometers in order to achieve particle transport in a reasonable period of time. The width and depth of the laminar flow channel and outlet channels must be large enough to allow passage of the desired particles, and any other particles associated with them, such as adsorbent or absorbent particles, and are preferably between about 2 or 3 times the diameter of any absorbent or adsorbent particles present in the streams and less than or equal to 5 mm. If the width dimension is in the wafer thickness direction, then for the silicon microfabricated embodiments of the microscale devices of the present invention, the width of the channels, inlets and outlets is less than the silicon wafer thickness, i.e. about 300 micrometers.
If the depth dimension is in the wafer thickness direction, then for the silicon microfabricated embodiments of the microscale extraction devices of the present invention the depth of the inlet and outlet is less than the silicon wafer thickness, i.e. about 300 micrometers. Preferably the depth, particularly of the laminar flow channel, is less than about 200 micrometers, and more preferably less than about 100 micrometers.
The laminar flow channel (called the xe2x80x9cextraction channelxe2x80x9d in the separation embodiment) receives the inflow of the sample and extraction streams from the sample and extraction stream inlets and conducts these streams in parallel laminar flow for a distance sufficient to allow extraction of the desired particles into the extraction stream. In the diffusion analysis embodiment of this invention, the laminar flow channel is long enough to permit small analyte particles to diffuse from the sample stream and have a detectable effect on an indicator substance or detection means, preferably at least about 2 mm long. The length of the flow channel depends on its geometry. The diffusion coefficient of the analyte, which is usually inversely proportional to the size of the analyte, affects the desired flow channel length. For a given flow speed, particles with smaller diffusion coefficients require a longer flow channel to have time to diffuse into the indicator stream.
The laminar flow channel can be straight or non-straight, i.e., convoluted. A convoluted flow channel as used herein refers to a flow channel which is not straight. A convoluted channel can be, for example, coiled in a spiral shape or comprise one or a plurality of xe2x80x9chairpinxe2x80x9d curves, yielding a square wave shape. Convoluted channels provide longer distances for diffusion to occur, thereby allowing for measurement of analytes with larger diffusion coefficients, e.g., typically larger analytes. In preferred embodiments of this invention wherein a silicon microchip is the substrate plate in which the flow channel is formed, the channel length of a straight flow channel is between about 5 mm and about 50 mm. In preferred embodiments of this invention wherein the flow channel is convoluted, i.e., non-straight, the length of the flow channel is defined or limited only by the size of the microchip or other substrate plate into which the channel is etched or otherwise formed. The channel width (diffusion direction) is preferably between about 20 micrometers and about 1 mm. The channel is more preferably made relatively wide, e.g. at least about 200 micrometers, which makes it easier to measure indicator fluorescence with simple optics, and less likely for particles to clog the channel. However, the channel can be made as narrow as possible while avoiding clogging the channel with the particles being used. Narrowing the width of the channel makes diffusion occur more rapidly, and thus detection can be done more rapidly. The channel depth is small enough to allow laminar flow of two streams therein, preferably no greater than about 1000 micrometers and more preferably between about 50 micrometers and about 400 micrometers.
In some embodiments of the diffusion analysis invention, the laminar flow channel may be long enough to allow the indicator and sample streams to reach equilibrium with respect to the analyte particles within the channel. Equilibrium occurs when the maximum amount of smaller particles have diffused into the indicator stream.
Alternatively, to allow more time for diffusion to occur, the flow rate can be decreased. However, several factors limit the minimum flow rate and therefore make a longer flow channel desirable in some cases. First, the flow rate is achieved by a pumping means or pressure source, some of which cannot produce as low a pressure and flow rate as may be desired, to allow enough time for diffusion of particles with small diffusion coefficients. Second, if the flow rate is slow enough and some particles are of significantly different density from the surrounding fluid streams, particles denser than the surrounding fluid streams may sink to the bottom of the flow channel and particles less dense than the surrounding fluid streams may float to the top of the flow channel. It is preferable that the flow rate be fast enough that hydrodynamic forces substantially prevent particles from sticking to the bottom, top, or walls of the flow channel. Third, a small change in pressure leads to larger errors in measurement accuracy at lower flow rates. Fourth, at low flow rates, other factors, such as changes in viscosity of fluids, can lead to larger errors in measurement accuracy.
The inlet and outlet channels are preferably between about 2 to 3 times the maximum-sized stream particulate diameter and about 100 micrometers in width and between about 2 to 3 times the diameter of the maximum-sized particles and less than about 100 micrometers in depth, and the extraction channel is between about 2 to 3 times the diameter of the maximum-sized particles and about ⅔ the wafer thickness in width, between about 2 to 3 times the diameter of the maximum-sized particles and less than about 100 micrometers in depth, and between about 4 and about 10 times the diameter of the maximum-sized particles and less than or equal to 5 mm long.
In an embodiment of the separation device in which the particle transport direction is rotated 90 degrees from that of the xe2x80x9cHxe2x80x9d design, called the xe2x80x9cflat extraction devicexe2x80x9d herein, the inlet channels have a width equal to the extraction channel width at the entrance to the extraction channel of preferably between 2 and 3 particle diameters and about 500 micrometers, and the extraction channel is preferably between about 2 and 3 times the diameter of maximum-sized particles and less than or equal to 5 mm in width, between about 2 and 3 times the diameter of the maximum-sized particles and less than about 100 micrometers in depth, and between about 4 and about 10 times the diameter of the maximum-sized particles and less than or equal to 5 mm long.
The term xe2x80x9caspect ratioxe2x80x9d as used herein refers to the ratio of the width to the depth of a channel.
The laminar flow channels of this invention preferably have an aspect ratio less than 50. The aspect ratio may be less than 25 or any number from less than 1 to 49. Microfabricated devices of this invention which can be manufactured with laminar flow channels having aspect ratios less than 50 and having depths less than 100 micrometers have numerous advantages over similar constructions with larger aspect ratios and larger laminar flow channel depths. Motive forces on particles capable of effecting differential transport of desired particles within the laminar flow channel are the result of local field gradients. Ultra-small transport distances enable differential transport of desired particles faster than undesired particles in short periods of time, allowing for significant minimization of the size needed for the device at moderate extraction channel flow rates. In addition lower flow rates can be used.
Devices within the size range described above yield distinctive advantages when evaluated in the following performance categories: (a) power consumption to achieve objective, (b) size of device required to achieve the objective, and (c) integratability of devices in a plurality of systems for management and processing of very small fluid volumes in a batch (sample to sample) mode.
Some fields known to the art which may be used for differential transport of the particles in the devices of this invention are those produced by:
Sedimentation
Electrical energy
Temperature gradients
Cross Flow
Dielectrical gradients
Shear forces
Magnetic forces
Concentration gradients
Means for producing such fields are known to the art in connection with mesoscale and macroscale devices.
Because of the small sizes of the channels described herein, differential transport of desired particles by diffusion or other means occurs extremely rapidly, e.g. within less than about 300 seconds, and if desired, less than about one second. Devices according to this invention can be fabricated which will detect the presence or determine the concentration of desired or undesired particles in the product and/or by-product streams where these particles occur in less than five minutes, or if desired in less than four minutes, or less than three minutes, or less than two minutes, or less than one minute, or less than ten seconds, or less than one second.
In the microfabricated devices of this invention in comparison to the larger-scale devices of the prior art having channel depths greater than 100 micrometers, samples of much smaller size, e.g. about 1 mL, and down to about 1 picoliter, may be treated, whereas in larger devices, very small samples could be absorbed onto the channel walls. In addition, low Reynolds numbers for the flow are achieved, allowing for laminar flow and minimizing or totally eliminating turbulence which would interfere with differential extraction of desired particles.
In the separation embodiment of this invention, a portion of the desired particles in the sample stream (having larger diffusion coefficients than the undesired particles, or being more susceptible than the undesired particles to transport into the extraction stream when differential transport means are applied to the system) is transported to the product stream. When the extraction is diffusion-based, some of the smaller particles will always remain in the sample stream; however, the percentage of desired particles transported to the product stream can be increased by increasing the time of contact of the sample and extraction streams, e.g. by increasing the length of the extraction channel or reducing the flow velocity. For simple diffusion systems, the process may be timed such that the two streams are in contact up to the point where the concentration of smaller particles in both streams is almost equal.
The sample and extraction streams may have different properties e.g. viscosities, densities, surface energies, diffusion coefficients, homogeneities, chemical compositions and the like, which may affect the differential transport rates. System parameters may need to be adjusted and optimized to take account of these differing properties, as will be apparent to those skilled in the art.
The sample and extraction streams are kept in contact in the extraction channel for a period of time sufficient to allow an analyzable quantity of desired particles to be transported into the extraction stream. The amount of product recovered from the device may be between about 0.001 picoliter/sec and about 50 microliters/sec or more. For example, illustrated herein is an optimal flow rate for the product stream of about 200 nanoliters/sec. As is known in the art, even the very small amounts of analytes present in such small product streams may be detected by spectroscopic and other means.
Successful operation of the inventions described herein requires precise control of volume flow rates on three of the four channels of the device (i.e. sample, extraction, product, and by-product streams). The fourth channel need not and should not be regulated, as leaving this channel unregulated will allow the device to accommodate unpredictable changes in volume of the sample because of xcex94V of mixing of the sample and extraction streams. Means for achieving precisely regulated flow rates are known to the art. For devices with more or less inputs and outputs, flow control means connected to all but one of these are preferred.
To aid in controlling the size of particles being transported to the product stream in a diffusion-based extraction system of this invention, and reduce the appearance of larger particles in the product stream, a fluid barrier may be created in the extraction channel. Such a fluid barrier is present when the extraction stream is present in sufficient volume to cause a portion of the extraction stream to flow through the by-product exit with the exiting by-product stream, as illustrated in FIG. 3. Smaller particles diffusing into the extraction stream must cross the width of this fluid barrier before being able to exit with the product stream. Such fluid barriers formed on a larger scale are discussed in Williams P. S., et al. (1992), xe2x80x9cContinuous SPLITT Fractionation Based on a Diffusion Mechanism,xe2x80x9d Ind. Eng. Chem. Res. 2172-2181, incorporated herein by reference.
By controlling the pressure of the sample and extraction streams, the ratio of volume from each that enters the extraction channel can be controlled. The volume ratio of the sample stream and the extraction stream can also be set by the geometry of the outlet and inlet channels for a fixed delivery pressure on the sample and extraction streams. The volume flow rate of the product and by-product streams may also be controlled by manipulating the product and by-product stream pressures or by using arbitrary port (inlet) pressures and altering the flow resistance of the inlets. Whatever the control mode, the inlet and outlet channels must satisfy the criteria for minimum channel dimensions based on the size of the particulate to be processed as described above. If the volume of the extraction stream entering the extraction channel is greater than the volume of the sample stream, and the two exit streams are identical, a fluid barrier is formed. If the volume flow rate of the product stream is too small to accommodate the entire volume flow of the extraction stream then a fluid barrier will also be formed.
The diffusion analysis system of this invention, in addition to the sample and indicator stream inlet channels and the outlet channels, may also comprise specimen channels and outlet means such as smaller channels for conducting specimen streams from the indicator stream at successive intervals along the length of the laminar flow channel, and means including viewports and fluorescence detectors for measuring changes in an indicator substance in each specimen stream, whereby concentration of the analyte in the sample stream may be determined.
Dual detection embodiments of the device of the present invention which allow for detection of both undissolved and dissolved analytes are also provided. Detection of both undissolved and dissolved analytes can be achieved in one dual detection device: dissolved particles can be detected in the flow channel of the T-sensor and undissolved particles can be detected in a v-groove channel or sheath flow module, either or both of which can be in fluid connection with a T-sensor flow channel. Branching flow channels can provide for fluid connection between a T-sensor flow channel and a v-groove channel and/or sheath flow module.
The channel cell systems of this invention can be in fluid connection with a v-groove flow channel, which preferably has a width at the top small enough to force the particles into single file but large enough to pass the largest particles without clogging. V-groove channels are formed by anisotropic EPW (ethylenediamine-pyrocatechol-water) etching of single crystalline silicon microchips, providing access to reflective surfaces with precisely etched angles relative to the surface of the microchip (Petersen, Proc. IEEE 70(5): 420-457,1982). (U.S. patent application Ser. No. 08/534,515 (U.S. Pat. No. 5,726,751), xe2x80x9cSilicon Microchannel Optical Flow Cytometer,xe2x80x9d which is incorporated by reference herein in its entirety, discloses a flow cytometer comprising a v-groove flow channel formed by micromachining a silicon microchip.) The cross-section of such a channel is like a letter V, and thus is referred to as a v-groove channel. An optical head comprising a laser and small and large angle photodetectors adapted for use with a v-groove flow channel can be employed as well. As described in U.S. patent application Ser. No. 08/534,515 (U.S. Pat. No. 5,726,751), detectors placed at small and large angles with respect to the portion of the probe beam reflected from the v-groove wall can be used to count particles, such as cells, and distinguish them by size (via small angle detector) and structure/morphology (via large angle detector). Using an appropriate laser or LED source, e.g., a blue laser, which can be determined by routine choice by those of ordinary skill in the art, fluorescence detection can be performed by placing an appropriate filter in front of the large angle detector.
The laminar flow channel can be in fluid connection with a v-groove channel allowing for dual detection of dissolved and undissolved, single-file particles with one device. The fluid streams can flow first through a T-sensor flow channel and then through a v-groove channel, viabranching flow channels. Alternatively, the fluid stream can flow first through a v-groove channel and then through a laminar flow channel, e.g., of a diffusion analysis device of this invention.
An alternative means of achieving single file particle flow through a flow channel is the sheath flow module disclosed in U.S. patent application Ser. No. 08/823,747, xe2x80x9cDevice and Method for 3-Dimensional Alignment of Particles in Microfabricated Flow Channels,xe2x80x9d filed Mar. 26, 1997, now U.S. Pat. No. 6,159,739 issued Dec. 12, 2000, and specifically incorporated in its entirety by reference herein. The sheath flow module includes a first plate of material having formed therein a laminar fluid flow channel; at least two inlets, each inlet joining the laminar flow channel at a junction, the first inlet junction being wider than the second inlet junction, and an outlet from the flow channel. A second plate, e.g., a transparent cover plate, seals the module and allows for optical measurements. A transparent cover plate allows for optical measurements by reflection, in cases where the first plate is a reflective material, e.g., silicon. A first inlet allows for introduction of a first fluid into the flow channel. The first fluid is the sheath fluid. A second inlet allows for introduction of a second fluid into the sheath fluid while it is flowing through the flow channel. The second fluid is the center fluid. Because the second inlet junction is narrower than the first inlet junction, the center fluid becomes surrounded on both sides by the sheath fluid. After all fluids have been introduced and sheath flow has been achieved, the depth of the flow channel can be decreased, leading to vertical hydrodynamic focusing. Optionally, the width of the flow channel can be decreased, leading to horizontal hydrodynamic focusing. The decrease in depth and width can be gradual or abrupt Hydrodynamic focusing in the sheath flow module leads to single file particle flow.
The sheath flow module can be in fluid connection with the channel cell system of the present invention. The fluid streams can flow first through a T-sensor flow channel and then through a sheath flow module. Alternatively, the fluid stream can flow first through a sheath flow module and then through a T-sensor flow channel.
Channel cells of this invention may include multiple inlet branches in fluid connection with the laminar flow channel for conducting a plurality of inlet streams into said channel. These may be arranged in a xe2x80x9ccandelabraxe2x80x9d-like array or may be arranged successively along a xe2x80x9ccrossbarxe2x80x9d for the xe2x80x9cT,xe2x80x9d the branches of the xe2x80x9cY,xe2x80x9d or the inlet bar of the xe2x80x9cHxe2x80x9d configuration, the only constraint being that laminar flow of all the streams must be preserved.
Inlet means include the inlet channels or xe2x80x9cbranchesxe2x80x9d and may also include other means such as tubes, syringes, and the like which provide means for injecting feed fluid into the device. Outlet means include collection ports, and/or means for removing fluid from the outlet, including receptacles for the fluid, means inducing flow by capillary action, pressure, gravity, and other means known to the art. Such receptacles may be part of an analytical or detection device.
Embodiments of the devices of the present invention which allow for optical measurements in transmission are provided. In such embodiments, the channel cell system, or at least an analyte detection area, may transect the width of the substrate plate in which the channel cell system is formed. Substrate plate as used herein refers to the piece of material in which the channel cell system of this invention is formed, e.g., a silicon wafer and a plastic sheet. The analyte detection area, and optionally other parts of the channel cell system, lie between optically transparent plates in a space which cuts through the entire width of the substrate plate. Analyte detection area as used herein refers to that portion of the indicator stream where analyte particles create a detectable change in the indicator stream.
Optical measurements exploiting reflected light are referred to herein as detection by reflection, whereas optical measurements exploiting transmitted light are referred to herein as detection by transmission.
Extraction devices of this invention may comprise means for controlling the volume of extraction stream in the extraction channel with respect to the volume of the sample stream, which means include a product stream outlet smaller than required to allow the entire extraction stream to exit coupled with a by-product stream outlet large enough to handle the excess extraction stream. Extraction devices of this invention may comprise multiple product stream outlets so that product streams comprising different types of desired particles may be recovered.
The separation devices of this invention may be utilized as a sample pretreatment system for an analytical system including sensing means for detecting desired particles in the product stream. Such means include means for mixing the product stream with an indicator stream which interacts with the desired particles so as to allow them to be detected by sensing means known to the art, including optical means, such as optical spectroscopic equipment, and other means such as absorption spectroscopic equipment or means for detecting fluorescence, chemical indicators which change color or other properties when exposed to the desired particles of analyte, immunological means, electrical means, e.g. electrodes inserted into the device, electrochemical means, radioactive means, or virtually any microanalytical technique known to the art including magnetic resonance equipment or other means known to the art to detect the presence of analyte particles such as ions, molecules, polymers, viruses, DNA sequences, antigens, microorganisms, or other factors. Preferably, optical or fluorescent means are used, and antibodies, DNA sequences and the like are attached to fluorescent markers. Indicators and microfabricated mixing means, as well as detection and sensing means are described, e.g in copending application Ser. No. 08/625,808 (U.S. Pat. No. 5,716,852) incorporated herein by reference.
In a preferred embodiment of this invention the differential extraction device described above is integrated into an analytical system comprising means for further processing the product and/or by-product streams, such as diffusion-based mixing devices for mixing the product stream with an indicator substance (e.g. the T-sensor device), and detection chambers wherein the presence of desired analyte particles may be detected. These additional processing means are preferably incorporated with the differential extraction device in a xe2x80x9clab-on-a-chip,xe2x80x9d fabricated on a standard silicon wafer. In a preferred embodiment, the system comprises quantitation means for determining the concentration of the analyte particles (desired or undesired particles) in the product and/or by-product stream and/or determining the concentration of the analyte particles in the sample stream. Such means include spectroscopic equipment, potentiometric, amperometric, and dielectric relaxation equipment. Concentration determinations can be made by calculation or calibration by means known to the art and disclosed herein.
The differential extraction devices of this invention are used in a method for extraction of at least a portion of desired particles from a sample stream comprising said desired particles and also containing undesired particles, comprising: introducing said sample stream into the sample stream inlet of a microfabricated extraction device as described above; introducing an extraction stream into the extraction channel of said extraction device; and withdrawing a product stream comprising desired particles from the product stream outlet of said device.
Methods are also provided for detecting the presence of analyte particles in a sample stream, preferably a liquid stream, also comprising larger particles comprising: conducting said sample stream into a laminar flow channel; conducting an indicator stream, said indicator stream preferably comprising an indicator substance which indicates the presence of said analyte particles, by a detectable change in property when contacted with particles of said analyte into said laminar flow channel, whereby said sample stream and said indicator stream flow in adjacent laminar streams in said channel; allowing analyte particles to diffuse into said indicator stream; and detecting the presence of particles of the analyte in said indicator stream.
The methods are performed in either batch or continuous mode operation. In batch mode, sample sizes may be as small as about one picoliter, preferably no more than about 250 microliters and more preferably are no more than about 50 microliters, although sample sizes of up to 1 mL or 10 mL or greater are also contemplated. The methods are completed in a time period from less than 1 second to no more than about 5 minutes, although, again, the device can be fabricated to allow batch processing times of 10, 30, or 45 seconds, or 1, 2, 3 or 4 minutes, or less.
The batch method includes a start-up transition period wherein the fluid (which may be a gas) present within the device is displaced by the extraction and sample indicator and analyte streams as they enter the laminar flow channel until such time as the streams exist in a nearly equilibrium mass transport state.
A diffusion period follows during which time the streams are in contact in the laminar flow channel for a period of time sufficient to allow sufficient desired particles to be differentially transported into the other stream for analysis or further processing.
A shut-down device flush period then may be required during which a cleansing fluid such as water (or soap solution) or air or sequential combinations of water (or soap solution) and air is cycled through the device to remove both desired and undesired particles which may have been retained on the surface of the device.
The batch method of the separation embodiment of this invention which involves processing of one single, discrete sample at a time, may include recycle of the by-product stream into the sample stream inlet and repetition of the process to increase the amount of desired particles removed from the original sample. In this embodiment a sample of the undesired particles is generated which may be useful for subsequent analysis. The processes of this invention can be repeated until the desired particles have been substantially completely extracted from the sample stream.
In the continuous mode of this invention, the process may be continued for periods greater than 5 minutes. The steady state nature of this method makes longer signal integration times possible in the diffusion analysis embodiment.
Multiple devices of the separation embodiment of this invention can be arranged in series for the continuous mode so that the by-product stream from each device becomes the incoming sample stream to the next. This continuous application produces a series of finely regulated dilutions of the desired particles as well as a substantially clean stream of undesired particles upon exit from the last device of the series. In such an embodiment, the clean undesired particle by-product stream may also be routed to detection elements of the type mentioned above or to particulate sorting devices, counters, or sizing elements, such as a Si microfabricated flow cytometer, e.g. a silicon-based v-groove flow cytometer as described in U.S. patent application Ser. No. 08/534,515 filed Sep. 27, 1995, now U.S. Pat. No. 5,726,751; and 08/621,170 filed Mar. 20, 1996, now U.S. Pat. No. 5,747,349 issued May 5, 1998, incorporated herein by reference, or for further use. For example, in continuous mode, the devices of this invention may be used for dialysis, and the clear plasma stream recycled to a patients body.
The flow rate of the input streams is preferably between about 5 micrometers/second and about 5000 micrometers/second, more preferably about 25 micrometers/second. Preferably the flow rate for both streams is the same.
The methods and systems of the diffusion analysis embodiment of this invention include determining the concentration of the analyte particles in the sample stream by detecting the position within the laminar flow channel of analyte particles from the sample stream diffusing into the indicator stream causing a detectable change in the indicator stream or in an indicator substance in the indicator stream. The sample stream and the indicator stream may be allowed to reach equilibrium within the laminar flow channel. The location of the boundary of the detection area (i.e. that portion of the indicator stream containing diffused particles at a detectable concentration) with the unaffected indicator stream may be used to provide information about flow speed and/or sample concentration. The physical location of this boundary in the channel for a given analyte stays the same over time as long as the flow speed is constant and the sample unchanged. The location and size of the detection area can be varied by varying flow rate, sample concentration, and/or concentration of an indicator substance so as to optimize the signal for detection.
Information useful for determining the concentration of the analyte particles in the sample stream may be obtained by providing means for conducting specimen streams from the indicator stream at successive intervals along the length of the laminar flow channel, such as smaller channels equipped with viewports as described herein. Detection means such as those listed above are used to measure signals from the indicator stream. Changes in the intensity of the signals from specimen channel to specimen channel may be used to calculate the concentration of analyte particles in the original sample.
Advantages of the diffusion analysis system include the fact that analytes can be determined optically in turbid and strongly colored solutions such as blood without the need for prior filtering or centrifugation; cross-sensitivities of indicator dyes to larger sample components (a common problem) can be avoided; and the indicator can be kept in a solution in which it displays its optimal characteristics (e. g., cross-sensitivities to pH or ionic strength can be suppressed by using strongly buffered solutions). Measurements of the indicator stream at several locations along the channel can compensate for some remaining cross-sensitivities. In addition, the flow channel can be wide, which makes it easy to measure the indicator fluorescence with simple optics. No membrane is needed; the system is less subject to biofouling and clogging than membrane systems. The system is also tunable in that sample or indicator stream concentrations and/or flow rates can be varied to optimize the signal being detected. For example, if a reaction takes about five seconds, the system can be adjusted so that the reaction will be seen in the central portion of the device.
The sample stream may contain particles larger than the analyte particles which are also sensitive to the indicator substance. These do not diffuse into the indicator stream and thus do not interfere with detection of the analyte.
Additionally, a method for determining kinetic rate constants as a function of distance traveled by the input streams from the joint where the two streams meet is provided. Generally, kinetic measurements are made by plotting a physical property related to concentration versus time, i.e., time of reaction. The method provided herein for making kinetic measurements as a function of distance traveled by the sample and indicator stream, rather than as a function of time, is advantageous for the following reasons. The constituents of the streams, i.e., the particles, and the concentrations thereof, at a given position in the flow channel remain constant, given that the flow rate is constant. This method allows for integrating the data from detection, e.g., optical measurements, over time, thereby increasing the accuracy of the data collected and hence of the calculated/determined rate constants. Furthermore, if an experimental error occurs during detection, e.g. in the collection of data, at a given time, one can merely perform the detection measurement again, at the distance/position in the flow channel where the error occurred. In prior art methods of making kinetic measurements, if data at a given time point are lost due to experimental error, those data cannot be collected again during the same experiment.
Further applications of the principles set forth above are described in U.S. patent application Ser. No. 08/656,155 filed May 31, 1996 (now U.S. Pat. No. 5,726,404), U.S. patent application Ser. No. 60/000,281 filed Jun. 16, 1995, and U.S. patent application Ser. No. 08/665,218 filed Jun. 14, 1996 (now U.S. Pat. No. 5,922,210), U.S. patent application Ser. No. 08/621,170 filed Mar. 20, 1996 (now U.S. Pat. No. 5,747,349), U.S. patent application Ser. No. 08/534,515 filed Sep. 27, 1995 (now U.S. Pat. No. 5,726,571), U.S. patent application Ser. No. 08/736,336 filed Oct. 23, 1996 (now U.S. Pat. No. 5,748,827), U.S. patent application Ser. No. 08/823,747 filed Mar. 26, 1997 (now U.S. Pat. No. 6,159,739), U.S. patent application Ser. No. 08/876,038 filed Jun. 13, 1997 (now U.S. Pat. No. 5,971,158), U.S. patent application Ser. No. 08/938,584 filed Sep. 26, 1997, (now U.S. Pat. No. 6,136,272), U.S. patent application Ser. No. 08/938,093 filed Sep. 26, 1997 (now U.S. Pat. No. 6,007,775), U.S. patent application Ser. No. 08/961,345 filed Oct. 30, 1997 (now U.S. Pat. No. 5,974,867), U.S. patent application Ser. No. 08/938,585 filed Sep. 26, 1997, U.S. patent application Ser. No. 08/900,926 filed Jul. 25, 1997 (now U.S. Pat. No. 5,948,684), U.S. patent application Ser. No. 09/080,691 filed May 18, 1998, U.S. patent application Ser. No. 60/067,082 filed Nov. 20, 1997, U.S. patent application Ser. No. 09/196,473 filed Nov. 19, 1998, U.S. patent application Ser. No. 09/169,533 filed Oct. 9, 1998 (now U.S. Pat. No. 6,067,157), U.S. patent application Ser. No. 60/135,417 filed May 21, 1999, U.S. patent application Ser. No. 09/503,553 filed Feb. 14, 2000, U.S. patent application Ser. No. 09/574,797 filed May 19, 2000, U.S. patent application Ser. No. 60/137,386 filed Jun. 3, 1999, and U.S. patent application Ser. No. 09/579,666 filed May 26, 2000, all of which are incorporated herein by reference to the extent not inconsistent herewith.